Proliferation Of Human Lens Epithelial Cells Research Articles

Qiju Dihuang Pill protects the lens epithelial cells via alleviating cuproptosis in diabetic cataract

Ethnopharmacological relevanceQiju Dihuang Pill (QDP) is a traditional Chinese medicine prescription for the treatment of eye diseases. Novel literature reports that copper-induced cell death, called as cuproptosis, is a copper-dependent and differs distinctly from other types of cell death. Aim of the studyThe present study aims to investigate whether QDP could protect lens epithelial cells via alleviating copper-induced death in diabetic cataract. Materials and methodsThe different concentration of QDP medicated serum was administrated on high glucose (HG)-induced human lens epithelial cells (HLECs). The copper concentration was tested using Elabscience Copper Assay kit. The proliferation was detected using CCK-8 and EdU assays. The molecular binding was identified using RIP-PCR and luciferase reporter assay. ResultsResults indicated that HG culture condition triggered the copper concentration and repressed the proliferation of HLECs. Then, the elesclomol-Cu (Es–Cu) administration up-regulated the copper concentration and inhibited the proliferation, and cuproptosis inhibitor tetrathiomolybdate (TTM) could specifically reverse the consequence. QDP treatment reduced the copper concentration and cuproptosis-related genes (SLC31A1, FDX1). MeRIP-Seq and RIP-PCR confirmed that QDP reduced the stability of SLC31A1 mRNA through m6A modified site, and copper actually synergized the molecular binding efficiency. Rescue assay verified the role of QDP and SLC31A1 on HLECs’ cuproptosis characteristic. ConclusionThis research identified the protective role of QDP on HG-induced HLECs in DC through decreasing m6A/SLC31A1-mediated cuproptosis in DC. This finding provides novel insights into mechanisms for QDP and sheds light on the multifaceted role of traditional prescription on DC.

Knockout of TGF-β receptor II by CRISPR/Cas9 delays mesenchymal transition of Lens epithelium and posterior capsule opacification

Posterior capsule opacification (PCO) is the most common postoperative complication of cataract surgery. Transforming growth factor-β (TGF-β) is related to epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) that is proven to induce PCO formation in clinical and experimental studies. In this study, CRISPR sequences targeting exon of TGF-βRII were knocked out with lentiviral transfection in LECs. Rabbits' PCO model was established and recombinant adeno-associated virus (AAV) for transferring the gRNA of TGF βRII were intravitreally injected. SgRNA inhibited TGF-βRII expression and human LECs proliferation. In TGF-βRII knockout group, LECs motility and migration were suppressed, N-cadherin and vimentin expressions were significantly decreased, whereas E-cadherin was increased. The animal model showed that TGF-βRII knockout in vivo was effective in suppressing PCO. The current study suggested that the CRISPR/Cas9 endonuclease system could suppress TGF-βRII secretion, which participates in the EMT procedure of LECs in vitro and PCO in vivo. These findings might provide a new gene-editing approach and insight into a novel therapeutic strategy for PCO.

REPS2 downregulation facilitates FGF-induced adhesion and migration in human lens epithelial cells through FAK/Cdc42 signaling and contributes to posterior capsule opacification

Posterior capsular opacification (PCO) can cause postoperative visual loss after cataract surgery. Residual human lens epithelial cell (HLEC) proliferation, migration, epithelial-mesenchymal transition (EMT) and synthesis of extracellular matrix (ECM) are the entitative reasons for PCO. Low expression of Ral-binding protein 1-associated Eps domain-containing 2 (REPS2) and high levels of basic fibroblast growth factor (b-FGF) were observed in the lens and postoperative aqueous humor of cataract patients. REPS2 was identified as a negative regulator in growth factor signaling; however, its function in HLECs is unknown. This was first investigated in the present study by evaluating REPS2 expression in anterior lens capsules from cataract patients, a mouse cataract model, and HLE-b3 cells. The biological function of REPS2 in HLE-B3 cells was assessed by REPS2 silencing and Cell Counting Kit 8, wound healing, Transwell migration, F-actin staining, G-protein pulldown and western blot assays. In the present study, REPS2 was significantly downregulated in human and mouse cataract capsules and H2O2-treated HLE-B3 cells. REPS2 knockdown increased fibronectin, type I collagen, and α-smooth muscle actin expression levels and stimulated HLECs proliferation and migration; these effects were enhanced by FGF treatment and accompanied with focal adhesion kinase (FAK) phosphorylation, cell division cycle 42 (Cdc42) activation, focal adhesion protein upregulation, and F-actin cytoskeleton reorganization. However, treatment with the FAK inhibitor PF573228 abolished these effects. Thus, REPS2 downregulation in cataract HLECs induces their proliferation and facilitates FGF-induced ECM synthesis, EMT, cell adhesion and migration by activating FAK/Cdc42 signaling, which may underlie PCO pathogenesis.

Up-Regulation of GATA4 Regulates Human Lens Epithelial Cell Function in Age-Related Cataract

Purpose: GATA4 has emerged as a novel regulator that plays a critical role in mediating senescence. However, the role of GATA4 in age-related cataract (ARC), the leading cause of visual impairment, requires further elucidation. Methods: GATA4 expression was measured by quantitative RT-PCR and capillary Western immunoassay (WES). The MTT assay, EdU assay, and rhodamine-123/Hoechst and calcein-AM/propidium iodide double staining were used to investigate the role of GATA4 in the viability, proliferation, and apoptosis of cultured human lens epithelial cells (HLECs). Results: HLECs were subjected to 3 different treatment models, including prolonged exposure to low-dose H₂O₂, UVB irradiation, and mild heating, to simulate senescence and apoptosis. GATA4 expression was significantly increased in these models in a time- and dose-dependent manner. Overexpression of GATA4 reduced cell viability, accelerated apoptosis development, and reduced the proliferation of HLECs. Furthermore, the expression of GATA4 from ARC was up-regulated at both mRNA and at protein level compared with clear lenses. Conclusion: GATA4 is up-regulated in all 3 models of HLECs in vitro and the cells from ARC lenses in vivo. Up-regulation of GATA4 mediates HLEC dysfunction. GATA4-mediated effects in HLECs would provide a novel insight into the pathogenesis of ARC.

L‑carnitine alleviates oxidative stress‑related damage via MAPK signaling in human lens epithelial cells exposed to H2O2.

L‑carnitine (LC) is well known for its antioxidative properties. The present study aimed to evaluate the effects of LC on human lens epithelial cells (HLECs) and to analyze its regulatory mechanism in cataractogenesis. HLE B‑3 cells were cultured with hydrogen peroxide (H2O2) and were pretreated with or without LC. The Cell Counting kit‑8 assay was used to determine cell viability. Reactive oxygen species(ROS) assay kit was used to measure the cellular ROS production induced by H2O2 and LC. In addition, reverse transcription‑quantitative PCR and western blot analysis were performed to detect the expression levels of oxidative damage markers and antioxidant enzymes. Notably, ROS overproduction was observed upon exposure to H2O2, whereas LC supplementation markedly decreased ROS levels through activation of the antioxidant enzymes forkhead box O1, peroxiredoxin 4 and catalase. Furthermore, LC suppressed the expression of apoptosis‑associated genes (caspase-3) and inflammation‑associated genes [interleukin(IL)1, IL6, IL8 and cyclooxygenase‑2]. Conversely, LC promoted proliferating cell nuclear antigen, cyclin‑dependent kinase (CDK)2 and CDK4 expression, which may increase proliferation of HLECs that were incubated with H2O2. In addition, epithelial‑mesenchymal transition occurred upon ROS accumulation, whereas the effects of H2O2 on AQP1 and vimentin expression were reversed upon LC supplementation. Notably, this study revealed that LC restored the oxidant/antioxidant balance and protected against cell damage through the mitogen‑activated protein kinase signaling pathway. In conclusion, LC may serve a protective role in curbing oxidative damage and therefore may be considered a potential therapeutic agent for the treatment of cataracts.

MicroRNA-34a suppresses human lens epithelial cell proliferation and migration via downregulation of c-Met

MicroRNAs (miRNAs) are endogenously expressed, non-coding, small RNAs which inhibit protein translation through binding to target mRNAs. Recent studies have demonstrated that miRNAs participate in the regulation of a variety of cell structures and functions including those for cell proliferation and migration. MicroRNA-34a (miR-34a), a potential effector of the p53 tumor suppressor gene, is extensively studied for its suppression of cell growth. In the present study, we investigated the function of miR-34a in human lens epithelial cells. Following confirming that miR-34a expression was increased in a P53 dependent manner in human lens epithelial cells after treatment with doxorubicin, we demonstrated that overexpression of miR-34a in the human lens epithelial cell line HLE B3 led to a significant decrease in cell proliferation and migration, with the use of MTS and transwell migration assays. Moreover, HGF enhanced the proliferation and migration of human lens epithelial cells. miR-34a was found to downregulate the expression of c-Met protein by Western blotting. Furthermore, overexpression of miR-34a downregulated the levels of phosphorylated Akt, phosphorylated ERK1/2 and other cell cycle regulators. miR-34a expression was significantly reduced in posterior capsule opacification (PCO) clinical samples. These results demonstrate that miR-34a may act as a suppressor in PCO by regulating human lens epithelial cell proliferation and migration through downregulation of c-Met.

Circular RNA HIPK3 regulates human lens epithelial cells proliferation and apoptosis by targeting the miR-193a/CRYAA axis

Circular RNAs (circRNAs) are a novel class of non-coding RNAs generated from back splicing. Accumulating evidence has demonstrated their vital regulation in several biological processes and ocular diseases. However, the role of circRNAs in age-related cataract (ARC), the leading cause of visual impairment worldwide, is still unknown. CircRNA sequencing reveals that 101 circRNAs are differentially expressed between the capsules of transparent and ARC lenses, including 75 down-regulated circRNAs and 26 up-regulated circRNAs transcripts. Eight of 10 differentially expressed circRNAs are further verified by quantitative RT-PCRs. One highly conserved circRNA, circHIPK3, is significantly down-regulated in all cortical, nuclear and posterior subcapsular subtypes of ARC. The silencing of circHIPK3, but not HIPK3 mRNA, significantly accelerates apoptosis development upon oxidative stress and decreases cell viability and proliferation in primary cultured human lens epithelial cells (HLECs). The expression of α-SMA and vimentin was downregulated, while the expression of E-cadherin and ZO-1was upregulated, suggesting the repression of epithelial-mesenchymal transition after circHIPK3 knockdown. CircHIPK3 silencing increases miR-193a expression. miR-193a regulates CRYAA expression by targeting the binding site within the 3′UTR. Moreover, miR-193a decreases the viability and proliferation, and increases the apoptosis of HLECs upon oxidative stress. This study suggests that circRNAs are the potential regulators in cataractogenesis. CircHIPK3 regulates HLECs function through miR-193a-mediated CRYAA expression. This finding would provide a novel insight into the pathogenesis of ARC.

Sustained-release genistein from nanostructured lipid carrier suppresses human lens epithelial cell growth.

To design and investigate the efficacy of a modified nanostructured lipid carrier loaded with genistein (Gen-NLC) to inhibit human lens epithelial cells (HLECs) proliferation. Gen-NLC was made by melt emulsification method. The morphology, particle size (PS), zeta potentials (ZP), encapsulation efficiency (EE) and in vitro release were characterized. The inhibition effect of nanostructured lipid carrier (NLC), genistein (Gen) and Gen-NLC on HLECs proliferation was evaluated by cell counting kit-8 (CCK-8) assay, gene and protein expression of the proliferation marker Ki67 were evaluated with real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence analyses. The mean PS of Gen-NLC was 80.12±1.55 nm with a mean polydispersity index of 0.11±0.02. The mean ZP was -7.14±0.38 mV and the EE of Gen in the nanoparticles was 92.3%±0.73%. Transmission electron microscopy showed that Gen-NLC displayed spherical-shaped particles covered by an outer-layer structure. In vitro release experiments demonstrated a prolonged drug release for 72h. The CCK-8 assay results showed the NLC had no inhibitory effect on HLECs and Gen-NLC displayed a much more prominent inhibitory effect on cellular growth compared to Gen of the same concentration. The mRNA and protein expression of Ki67 in LECs decreased significantly in Gen-NLC group. Sustained drug release by Gen-NLCs may impede HLEC growth.

Changes of protein kinase regulatory pathway in inhibition of curcumin on human lens epithelial cells proliferation

Background Our previous study showed that curcumin suppresses the proliferation of human lens epithelial cells (LECs) in vitro and has an influence on the signal transduction of cyclic adenosine monophosphate (cAMP) and cyclic guanosinc monophosphate (cGMP). Actually, the regulation for biological behavior of LECs in vivo is complex. Objective This study was to investigate the changes of signal transduction of protein kinase (PK) in inhibition of curcumin on human LECs proliferation. Methods HLE-B3 was cultured and then divided into the blank control group, recombinant human basic fibroblast growth factor (rhbFGF) group and rhbFGF+ curcumin group.rhbFGF of 10 ng/ml was added in the medium in the rhbFGF group, and 20 mg/L curcumin was added into rhbFGF-induced cell medium for 24 hours in the rhbFGF+ curcumin group.The expression rates of PKA, PKC, PKG and calmodulin (CaM) in the cells were assayed using flow cytometry. Results The expression rates of PKA protein were (46.847±1.673)%, (33.250±2.242)% and (71.645±2.432)% in the blank control group, rhbFGF group and the rhbFGF+ curcumin group, respectively, and the expression rate of PKA protein was significantly reduced in the rhbFGF group compared with the blank control group (t=11.904, P<0.01), but the expression rate of PKA protein in the rhbFGF+ curcumin group was significnatly higher than that in the rhbFGF group (t=28.430, P<0.01). In the blank control group, rhbFGF group and the rhbFGF+ curcumin group, the expression rates of PKC protein in the cells were (35.575±1.937)%, (50.652±2.068)% and (27.662±4.481)%, those of PKG protein were (63.838±0.486)%, (86.562±1.325)% and (40.733±2.175)%, while those of CaM protein were (67.408±1.391)%, (83.887±3.499)% and (53.785±1.942)%, the expression rates of PKC, PKG and CaM were significantly lower in the rhbFGF group in comparison with the blank control group (all at P<0.01), and those in the rhbFGF+ curcumin group were significantly declined in comparison with the rhbFGF group (all at P<0.01). Conclusions Suppression of curcumin on HLE-B3 proliferation probably is associated with the up-regulation of PKA expression and down-regulation of PKC, PKG and CaM expression in the cells. Key words: Curcumin; Protein kinase; Signal transduction; Lens epithelial cells

Suppression and mechanism of zinc chloride on viability of human lens epithelial cells in vitro

Background Zinc ions can inhibit the proliferation of tumor cells and induce the apoptosis of cancer cells and commonly used in the topical treatment of conjunctivitis and trachoma. However, the effect of zinc ions on proliferation of human lens epithelial cells (LECs) is still unclear. Objective The purpose of the present study was to investigate the effect of ZnCl2 on human LECs proliferation and its mechanism. Methods Human LECs strains, HLEB-3 cells, were routinely cultured and passaged. Different concentrations of ZnCl2 was added into the medium to make the final concentrations to 5, 10, 20, 40 and 80 mg/L for 24, 48 and 72 hours, and the cultured cells without ZnCl2 were as the control group. Viability of the cells was detected by MTT assay; the mitochondrial membrane potential (ΔΨm) was measured using the JC-1 assay kit; the proportion of the cells in different cycles was detected using flow cytometry; meanwhile, the expressions of bcl-2 gene and bcl-2 protein were assayed by real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) technique, respectively. Results Cultured cells showed polygon-like with the tight arrangement in the control group. However, the number of the cells was obviously decreased with the irregular shape and disorder arrangement in different concentrations of ZnCl2 groups. The viability of the cells was significantly reduced with the increase of ZnCl2 concentrations and the lapse of active time (Fconcentrations=173.949, P=0.000; Ftime=16.997, P=0.000). The strong orange fluorescence in cellular membrane was seen in the control group, but the orange fluorescence was gradually weakened and green fluorescence was gradually enhanced with the increase of concentrations of ZnCl2. The cell proportions in S phase and G2/M phase were significantly raised and those in the G0/G1 phase were declined in different concentrations of ZnCl2 groups in comparison with the control group (S phase: F=15.594, P=0.000; G2/M phase: F=12.792, P=0.000; G0/G1 phase: F=43.366, P=0.000). The mean relative expression levels of bcl-2 mRNA in the cells were 1.00±0.00 in the control group, which were significantly higher than 0.32±0.13, 0.07±0.03, 0.14±0.05, 0.01±0.00 and 0.12±0.06 in the 5, 10, 20, 40 and 80 mg/L ZnCl2 groups (all at P<0.01). The expressing levels of bcl-2 protein in the cells were (138.57±32.80) pg/mg, which were significantly higher than (79.20±5.70), (79.95±6.21), (61.02±10.74), (72.05±11.61) and (55.97±10.53) pg/mg in the 5, 10, 20, 40 and 80 mg/L ZnCl2 groups (all at P<0.01). Conclusions ZnCl2 can arrest the HLEB-3 cells in G2/M phase and therefore suppresses the proliferation of HLEB-3 cells probably by reducing the ΔΨm and down-regulating the expression of bcl-2. Key words: Zinc ion; Lens; Epithelial cell; Bcl-2 gene; Cell proliferation

HSF4 Promotes Human Lens Epithelial Cells G1/S Arrest through Stabilizing p53

The differentiation from constantly dividing epithelial cells into fiber cells is the key step during lens development. Failure in lens fiber cells differentiation, which requires cell proliferation inhibition and cell cycle exit, causes cataract formation. It has been discovered that HSF4 (Heat Shock Factor 4) gene mutations led to congenital and senile cataracts. And the lens of HSF4 knockout mice showed increased proliferation of epithelial cells and premature differentiation of fiber cells. However, the molecular roles that HSF4 mutations play in cataract formation remain obscure. In present study, we found that wild type HSF4 suppressed human lens epithelial cells (HLECs) proliferation by promoting G1/S arrest. We further identified that HSF4 could interact with p53 and stabilize p53 protein by inhibiting its ubiquitin degradation.The p53 transcriptional activity was also increased by HSF4. In contrast, HSF4 cataract mutants had no effects on either HLECs proliferation or p53 protein stabilization. On the other hand, the wild type HSF4 lost its ability in cell proliferation inhibition and G1/S cell cycle arrest in p53‐null H1299 cell line. Our data reveals that HSF4 may work as a switch between lens epithelial cell proliferation and fiber cell differentiation, and p53 is required in this process.

Zinc oxide nanoparticles inhibit Ca2+-ATPase expression in human lens epithelial cells under UVB irradiation

Epidemiological and experimental studies have revealed that lens epithelial cells exposed to ultraviolet B (UVB) light could be induced apoptosis, and lens epithelial cell apoptosis can initiate cataractogenesis. Posterior capsular opacification (PCO), the most frequent complication after cataract surgery, is induced by the proliferation, differentiation, migration of lens epithelial cells. Thus, inhibiting the proliferation of lens epithelial cells could reduce the occurrence of PCO. It is reported that zinc oxide (ZnO) nanoparticles have great potential for the application of biomedical field including cancer treatment. In the present study, we investigated the cytotoxic effect of ZnO nanoparticles on human lens epithelial cell (HLEC) viability. In addition, changes in cell nuclei, apoptosis, reactive oxygen species and intracellular calcium ion levels were also investigated after cells treated with ZnO nanoparticles in the presence and absence of UVB irradiation. Meanwhile, the expression of plasma membrane calcium ATPase 1 (PMCA1) was also determined at gene and protein levels. The results indicate that ZnO nanoparticles and UVB irradiation have synergistic inhibitory effect on HLEC proliferation in a concentration-dependent manner. ZnO nanoparticles can increase the intracellular calcium ion level, disrupt the intracellular calcium homeostasis, and decrease the expression level of PMCA1. UVB irradiation can strengthen the effect of reduced expression of PMCA1, suggesting that both UVB irradiation and ZnO nanoparticles could exert inhibitory effect on HLECs via calcium-mediated signaling pathway. ZnO nanoparticles have great potential for the treatment of PCO under UVB irradiation.

Characterization of Zinc Oxide Nanoparticles and their Inhibitory Effect on Human Lens Epithelial Cells under UVB Irradiation

Posterior capsular opacification is the most frequent complication after cataract surgery, which is induced by the proliferation, differentiation and migration of lens epithelial cells. Inhibiting the proliferation of human lens epithelial cells could reduce the occurrence of posterior capsular opacification. To explore the possible potential of ZnO nanoparticles in treating posterior capsular opacification, we investigated the effect of ZnO nanoparticles on the human lens epithelial cell proliferation in the presence and absence of ultraviolet B (UVB) irradiation via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The results indicate that ZnO nanoparticles could apparently inhibit the proliferation of human lens epithelial cells in a dose-dependent manner, and UVB irradiation can further enhance this inhibitory role for the human lens epithelial cell proliferation, indicating the great potential of ZnO nanoparticles in treating posterior capsular opacification in clinical application.

Effect of long-term power frequency electromagnetic field exposure on proliferation and apoptosis of SRA01/04 cells

To investigate the effect of long-term power frequency electromagnetic field (50 Hz) exposure on the proliferation and apoptosis of human lens epithelial cells (SRA01/04 cells). SRA01/04 cells in the exponential growth phase were exposed or sham-exposed to power frequency electromagnetic field (50 Hz, 2.3 mT) for 2 hours per day, 5 days every week. After 11 weeks of exposure, the cells were collected; the cell morphology was observed under a microscope, the cell viability was measured by MTT assay, the cell cycle and apoptosis were examined by flow cytometry, and the protein expression levels of cyclin D and proliferating cell nuclear antigen (PCNA) were determined by western blot. Compared with the sham-exposed SRA01/04 cells, most exposed cells became rounded and more stereoscopic, and heterochromatin gathered near the nuclear membrane in some exposed cells. The MTT assay showed that the viability of exposed cells was significantly increased compared with that of the sham-exposed cells (P < 0.05). Long-term power frequency electromagnetic field exposure led to significantly increased number of cells in S phase (P < 0.05), and the proliferation index was significantly higher in the exposed cells than in the sham-exposed cells (P < 0.05). There was no significant difference in apoptotic rate between the exposed cells and sham-exposed cells (P > 0.05). The exposed cells had significantly higher protein expression levels of cyclin D and PCNA than the sham-exposed cells (P < 0.05). Long-term power frequency electromagnetic field exposure can promote cellular proliferation and change cell cycle in SRA01/04 cells, but it has no marked effect on the apoptosis of SRA01/04 cells.

EGF receptor inhibitor erlotinib as a potential pharmacological prophylaxis for posterior capsule opacification

Posterior capsule opacification (PCO) is the most frequent complication after cataract surgery, leading to a loss of sight if untreated. Erlotinib might be of therapeutic interest as an effective target agent (selective EGF-tyrosin-kinase-1 inhibitor). In this in-vitro study, erlotinib was evaluated for ocular biocompatibility and its effect on cell proliferation, migration, 3D matrix contraction and spreading of human lens epithelial cells. To exclude toxic concentrations, erlotinib was assessed for its biocompatibility on five different human ocular cell types in vitro by the tetrazolium dye-reduction assay (MTT) and the Live-Dead assay. To determine its effect on human lens epithelial cell (HLE-B3) proliferation, the MTT test was performed after incubation with different concentrations of erlotinib. Chemotactic migration was analyzed with the Boyden chamber assay and chemokinetic migration was assessed by time lapse microscopy. Contraction was measured by a 3D collagen type 1 matrix contraction assay, and cell spreading was determined by measuring the cell diameter on a fibronectin coated surface. The maximum non-toxic concentration of erlotinib was determined to be 100 μM in cell culture. Erlotinib potently inhibits human lens epithelial cell proliferation, with an IC50 of about 10 μM (8.8 μM ± 0.9 μM SD; r (2) =0.94). Chemotactic migration (p=0.004) and chemokinetic migration (p=0.001) were reduced significantly in a concentration-based manner. Erlotinib prevented human lens epithelial cells from matrix contraction (p=0.001) and cell-spreading (p=0.001). Erlotinib might become of clinical relevance for PCO prophylaxis in the future since it displayed good biocompatibility on ocular cells and mitigated human lens epithelial cell proliferation, migration, contraction, and spreading in vitro. Further studies are warranted to evaluate its potential for clinical application.

Effects of exogenous secreted protein acidic and rich in cysteine on human lens epithelial cell proliferation and adhesion

Many factors can impact human lens epithelium biological behavior. Secreted protein acidic and rich in cysteine (SPARC) is a newly discovered protein that can impact cell activity and may potentially affect human lens epithelial cells (HLECs). HLEC (SRA01/04) proliferation was detected by methyl thiazolyl tetrazolium (MTT) and the proliferation index was assessed by flow cytometry with SPARC at concentrations of 0, 0.2, 1 and 5 μg/ml. SPARC’s effect on cell cycle regulatory genes CCND1 and P21Waf1 was analyzed by PCR and SPARC’s effect on cell adhesion was detected by an enzyme labeling assay. SPARC displayed an inhibitory effect on HLEC proliferation. The amount of inhibition was related to SPARC concentrations and cell culture times. The inhibition rates were 9.67, 48.11 and 68.12% at concentrations of 0.2, 1 and 5 μg/ml, respectively, after 72 h of cell culture. With a MTT method, SPARC showed counter-adhesion of HLECs and this counter-adhesion was related to the concentration of SPARC. SPARC decreased the number of S+G2/M cells and caused G0/G1 phase arrest. With a 5 μg/ml SPARC treatment, CCND1 mRNA levels were significantly decreased and P21Waf1 mRNA levels were increased in comparison with the control group. SPARC inhibits HLEC proliferation and adhesion, which is related to the SPARC concentration and cell culture time. Therefore, SPARC seems to influence HLEC biological behavior. Key words: Secreted protein acidic and rich in cysteine (SPARC), human lens epithelial cells (HLEC), adhesion, proliferation, cell cycle regulatory gene.

Wnt3a-induced proliferation in human lens epithelial cell and its mechanism

Background Aberrant proliferation of residual lens epithelial cells (LECs) is one of main causes of posterior capsular opacification (PCO).Researches indicated that Wnt3a signaling pathway promote proliferation of epithelial cells,but its effect on LECs is still unclear. Objective The present study was to investigate the effects of Wnt3a on proliferation of human LECs and its mechanism and to provide a new gene target in the prevention and treatment of PCO. Methods Human LECs line (SRA01/04 cells ) was cultured and then incubated to 6-well plate at the density of 4×105/well.A human Wnt3a cDNA expressing vector targeted human LECs was constructed to increase the Wnt3a expression in SRA01/04 cells,and pcDNA3-HA expression vector was used as the control group.The expression of Wnt3a was identified by Western blot assay after transfected.The growth and proliferation of SRA01/04 cells were detected by MTT and flow cytometry (FCM).The expressions of β-catenin,cyclin D1 and c-myc in the cells were detected by Western blot assay.β-Catenin expression was localized using immunofluorescence assay,and the expression and localization of proliferating cell nuclear antigen ( PCNA ) were analyzed by immunocytochemistry for the exploration of the active mechanism of Wnt3a to proliferation of LECs.Results Human Wnt3a cDNA expression vector was designed successfully and transiently transfected to SRA01/04 cells,and Wnt3a/SRA01/04 cells and pcDNA3-HA/SRA01/04 cells were obtained.The expression of Wnt3a was verified in the Wnt3a transfected group compared with the control group.MTT indicated that the cell proliferating rate was significantly different between the Wnt3a transfected group and the control group ( Fgroup =15.235,P =0.005 ;Ftime =369.677,P =0.000),and that in various time points after transfected was significantly different (t =20.843,P=0.001 ;t =26.214,P＜0.001 ;t=25.177,P=0.001 ;t =35.516,P＜0.001 ;t =615.056,P＜0.001 ).The proportion of SRA01/04 cells in G1 phase was 51.74％ in the Wnt3a cDNA transfected group,with a significantly decrease in comparison with 79.44％ of the control group.However,the proportion of SRA01/04 cells in S phase in the Wnt3a cDNA transfected group was higher than that of the control group (36.23％ versus 12.34％ ).The positive expression rate of PCNA protein in SRA01/04 was (47.00％ ±7.58％ ) in the Wnt3a cDNA transfected group and ( 16.00％ ±3.61％ ) in the control group with a significant difference between them (t =8.256,P＜0.01 ).After 48 hours of transfection of the Wnt3a cDNA,the expression amount of β-catenin proteins was higher and the immunofluorescence was stronger in cell nucleus,and the expressions of cyclin D1 and c-myc proteins were elevated in Wnt3a/SRA01/04 cells. Conclusions The overexpression of Wnt3a activates the Wnt/β-catenin signaling pathway and downregulates the expression of a subset of target genes,including cyclin D1 and c-myc,which plays an important role in promoting the proliferation of human LECs. Key words: Wnt signal pathway; Lens epithelial cell; Cell proliferation; Posterior capsular opacification

Mechanisms of inhibition of elemene on human lens epithelial cell proliferation in vitro.

To study the effects of elemene (Ele) on proliferation and cell cycle of human lens epithelial cells B3 (HLE-B3) and the mechanisms of its signal transduction. Recombinant human basic fibroblast growth factor (rhbFGF) was used to induce proliferation of HLE-B3 cells, which were incubated with 80mg/L Ele for 24 hours. The inhibitory effects of Ele on the proliferation of HLE-B3 cells were evaluated by MTT method. The effect of Ele on HLE-B3 cell cycle was analyzed by flow cytometry(FCM). The expressions of protein kinase A (PKA) and protein kinase G (PKG) of HLE-B3 were also analyzed by FCM. Ele altered the cell cycle of HLE-B3 and effectively inhibited HLE-B3 cell proliferation induced by rhbFGF. Ele up-regulated PKA and down-regulated the expression of PKG in HLE-B3 cell. Ele inhibits HLE-B3 proliferation, making it an attractive potential agent in regimens to treat after-cataracts.

Influence of RGDRGD peptide on proliferation and adhesion of human lens epithelial cells in vitro

Background RGD is a small molecular multiple peptide containing Arg-Gly-Asp with an important role in inhibiting the adhesion,migration and neovascularization of tumor.Our previous study determined that RGD can suppress the adhesion and proliferation of lens epithelial cells(LECs),and RGDRGD may be of a stronger effect. Objective Present study was to investigate and compare the effect of RGDRGD peptide on the proliferation and adhesion of immortalized human LECs(HLEB-3)in vitro. Methods Human LECs harvested by trpsin-EDTA were suspended in DMEM medium with serial dilutions of RGD peptide and RGDRGD peptide(from 1000 mg/L to 250 mg/L)at 37℃ for 15 minutes as experimental group,and the HLECs cultured by common culture medium were used as the control group.The cells were then seeded into the 96-well plates with precoated fibmnectin (FN)and I collagen at the density of 2×104/ml.MTT stainingcolorimetry was used to measure the adhesion rates of lens epithelial cells cultured in different concentrations RGDRGD and RGD peptides after 1 hour.Cells were seeded into the 96-well plates for 24 hours at 37℃ in 5% CO2.Medium was then replaced with DMEM overnight.Subsequently,the cells were treated with serial dilutions of RGD and RGDRGD(from 2000 mg/L to 250 ms/L)dissolved in DMEM medium plus 20% fetal bovine serum.The inhibition of RGDRGD and RGD on the adhesion and proliferation of Human LECs was analyzed by MTT aher 24,48 and 72 hours. Results The inhibition rate of RGD peptide on the adhesion of LECs was gradually enhanced with the increase of concentration with the significant difference among the different concentrations groups(F=1089.56,P＜0.01),and the statistically significant elevation in inhibitory rate was found in RGDRGD peptide compared with RGD peptide(P＜0.01).The inhibition rate of RGDRGD peptide on the proliferation of LECs wag gradually increased with the increase of concentration with the significant difference among the different concentrations groups with a strongest effect in 1000 ms/L group(F=127.31,P＜0.01),and the much stronger inhibition Wag Been in RGDRGD peptide(F=1589.85,P＜0.01).The suppression rate of RGDRGD on LECs proliferation Wag much stronger with the prolong of time(F=1606.43,P＜0.01). Conclusion RGDRGD peptide and RGD peptide have inhibitory effect on adhesion and proliferation of human LECs in a dose-and time-dependent manner.Effect of RGDRGD peptide is much stronger than RGD peptide.These results imply that RGDRGD peptide and RGD peptide have the important role for prevention of PCO. Key words: RGDRGD peptide; Lens epithelial cells; Posterior capsular opacity

Inhibitory effects of NO-fluvastatin on proliferation of human lens epithelial cells in vitro by modulating cell cycle regulatory proteins

The effects of NO-Fluvastatin on proliferation of human lens epithelial cells (HLECs) and the action mechanism were investigated. Cell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. The expression of cell cycle regulatory proteins CyclinE mRNA and P21(waf1) mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). MTT staining colorimetry showed that HLECs proliferation was markedly inhibited by NO-Fluvastatin and the effect was dependently related to time (24, 48 and 72 h) and dosage (1, 5 and 20 mumol/L). Flow cytometry revealed that NO-Fluvastatin could significantly block HLECs in the G(0)/G(1) phase, resulting in the increased cells in the G(0)/G(1) phase and decreased in the S phase (P<0.05). RT-PCR showed that NO-Fluvastatin could obviously inhibit the CyclinE mRNA expression and induce the P21(waf1) mRNA expression as compared with the negative control groups (P<0.05). This experiment suggested that NO-Fluvastatin could suppress the proliferation of HLECs by regulating cell cycle regulatory proteins (inhibiting the expression of CyclinE mRNA and inducing the expression of P21(waf1) mRNA), resulting in the arrest of HLECs in the G(0)/G(1) phase, which can offer theory basis for NO-Fluvastatin in treating posterior capsular opacification in clinic practice.